1. RNAi Screen in Mouse Astrocytes Identifies Phosphatases that Regulate NF-κB Signaling 
Shitao Li, Lingyan Wang, Michael A. Berman, Ye Zhang, Martin E. Dorf 
Molecular Cell 
Volume 24, Issue 4, Pages (November 2006)
DOI: /j.molcel
Copyright © 2006 Elsevier Inc. Terms and Conditions

2. Figure 1
RNAi Screens of Phosphatase Genes that Regulate NF-κB Transcriptional Activity
(A) The design of the pBabe-Dual vectors. The positions of the puromycin resistance gene and the U6 and H1 promoters are indicated.
(B) Data from representative experiments depict the modulation of NF-κB reporter activity for each of the 250 phosphatase siRNA targets in non-TNF-treated astrocytes.
(C) The regulation of NF-κB reporter activity after TNFα stimulation of astrocytes transfected with each pair of siRNA targeting constructs. The luciferase activity of cells transfected with control siRNA vector with TNFα was set at 1. The activity of NF-κB signaling was quantified by measurement of the log ratio of firefly luciferase activity as standardized to that of Renilla luciferase. Dashed lines indicate two standard deviations (2 × SD).
(D) Knockdown of 13 phosphatases activated NF-κB-binding activity in CTFA. White bars represent responses of unstimulated astrocytes; shaded bars represent responses of TNFα-treated cells. Stimulation with TNFα triggered a 6- to 9-fold increase in CTFA activity; this value was normalized to 1 for comparison. Error bars represent the SD of three independent experiments.
(E) Knockdown of six phosphatases suppressed NF-κB-binding activity in CTFA assays. Error bars represent the SD of three independent experiments.
(F) The fold induction of NF-κB reporter activity after treatment with TNFα (shaded bars) or medium (open bars) in NIH3T3 cells transfected with the indicated pair of phosphatase siRNA targeting constructs.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2006 Elsevier Inc. Terms and Conditions

3. Figure 2
PP2A Catalytic and Structural Components Selectively Form Complexes with IKK and p65 NF-κB
(A) The indicated myc-tagged phosphatases were transfected into astrocytes and immunoprecipitated with either anti-myc or anti-IKKβ antibody. The 50 kDa bands were IgG heavy chain.
(B) Cell lysates were immunoprecipitated with anti-PP2A catalytic subunit (PP2A C) or anti-IKKβ antibody showing the interaction between endogenous IKK and PP2A. The 25 kDa and 50 kDa bands were IgG light and heavy chains.
(C) PPP2CB/PP2R1A core enzyme dephosphorylates Ser181 of IKKβ. Astrocytes were transfected with IKKβ. Ten minutes after TNFα stimulation, the lysates were immunoprecipitated with anti-IKKβ antibody. In a separate transfection, 3T3 cells were transfected with myc-tagged PPP2R1A plus Flag-tagged PPP2CB or other Flag-tagged phosphatases. The phosphatase components were eluted from the Sepharose beads with Flag peptide, and then 2 ng was incubated with immunoprecipitated IKKβ for 1 hr at 30°C and immunoblotted with anti-phospho-IKKβ antibody.
(D) PPP2CB RNAi did not synergize with the constitutively active IKKβ SSEE mutant to enhance NF-κB reporter activity.
(E) PPP2CB RNAi enhanced IKKβ phosphorylation. IKKβ-myc was cotransfected with PPP2CB RNAi or control RNAi into astrocytes. After TNFα stimulation, the lysates were harvested and immunoblotted with anti-IKKβ antibody and anti-phospho-IKKβ antibody.
(F) The indicated myc-tagged phosphatases were transfected into astrocytes and immunoprecipitated with either anti-myc or anti-NF-κB p65 antibody. The 50 kDa band is IgG heavy chain.
(G) Cell lysates were immunoprecipitated with anti-PP2A catalytic subunit (PP2A C) or anti-NF-κB p65 showing the interaction between endogenous NF-κB and PP2A. The 25 kDa and 50 kDa bands were IgG light and heavy chains.
(H) PPP2CA /PPP2R1B complexes dephosphorylate Ser536 of p65 NF-κB. Astrocytes were transfected with NF-κB p65. Ten minutes after TNFα stimulation, cells were harvested and immunoprecipitated with anti-NF-κB p65 antibody. In a separate transfection, 3T3 cells were transfected with the Flag-tagged PPP2CA and myc-tagged PPP2R1B or other Flag-tagged phosphatases. These phosphatases were eluted from the Sepharose beads with Flag peptide, and then 2 ng was incubated with immunoprecipitated NF-κB p65 for 1 hr at 30°C and immunoblotted with anti-p65 or two anti-phospho-NF-κB antibodies. The right panel depicts the protein levels of the indicated Flag-tagged phosphatases.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2006 Elsevier Inc. Terms and Conditions

4. Figure 3
Dephosphorylation of TRAF2 by PP2A Holoenzyme Inhibits NF-κB Activity
(A) The indicated myc-tagged phosphatases were transfected into astrocytes and immunoprecipitated with either anti-myc or anti-TRAF2 antibody.
(B) Cell lysates were immunoprecipitated with anti-PP2A catalytic subunit (PP2A C) or anti-TRAF2 showing the interaction between endogenous IKK and TRAF2. The 25 kD and 50 kD bands were IgG light and heavy chains.
(C) PPP2R5C-myc was cotransfected with Flag-tagged TRAF2 into astrocytes and immunoprecipitated with either anti-Flag or anti-myc antibody.
(D) Map of various TRAF2 constructs and their ability to associate with PPP2R5C.
(E) Indicated phosphatase genes were cotransfected with TRAF2 into astrocytes. Overexpression of PPP2R5C inhibited TRAF2-induced NF-κB reporter activity. Error bars represent the SD of three independent experiments.
(F) After IL-1β stimulation, PPP2R5C RNAi failed to stimulate NF-κB reporter activity in astrocytes.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2006 Elsevier Inc. Terms and Conditions

5. Figure 4 TNF-Induced TRAF2 Thr117 Phosphorylation
(A) Amino acid sequence alignment of a portion of the first zinc finger domain of TRAF2.
(B) The fold induction of NF-κB reporter activity in astrocytes transfected with TRAF2 and different Ser/Thr to Ala mutants.
(C) The fold induction of NF-κB reporter activity in TRAF2−/− MEFs transfected with TRAF2 and different Ser/Thr-to-Ala mutants. Error bars represent the SD of three independent experiments.
(D) The zinc finger domain (residues 99–271) showed two bands by electrophoresis in a 4%–20% SDS-PAGE gel. The upper band was sensitive to CIP phosphatase treatment.
(E) Thr117-to-Ala mutation abolished the upper band of the first finger domain.
(F) Specificity of anti-phospho-TRAF2 (Thr117) antibody.
(G) Time course of TNF-induced TRAF2 Thr117 phosphorylation.
(H) PPP2R5C inhibited Thr117 phosphorylation. Astrocytes were transfected Flag-TRAF2 with or without PPP2R5C-myc. Cells were treated with or without TNFα for 15 min before harvest and immunoprecipitation with anti-IKKβ or anti-NF-κB p65 antibodies and immunoblotted with the indicated antibodies.
(I) PPP2R5C RNAi enhanced TRAF2 phosphorylation. TRAF2-Flag was cotransfected with PPP2R5C RNAi or control RNAi into astrocytes. After TNFα stimulation, the lysates were harvested and immunoblotted with anti-Flag antibody and anti-phospho-TRAF2 (Thr117) antibody.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2006 Elsevier Inc. Terms and Conditions

6. Figure 5 Phosphatases Regulate Chemokine and Cytokine Expression
(A) The relative mRNA levels of MCP-1 (open bars), KC (shaded bars), and IL-6 (solid bars) in RNAi-transfected astrocytes without stimulation. Astrocytes were transfected with phosphatase RNAi vectors and cultured for 72 hr. The cDNAs were analyzed by real-time PCR. All phosphatase mRNA levels were normalized with the housekeeping gene β-glucuronidase. The dashed lines represent a 4-fold increase and 70% reduction, respectively. Error bars represent the SD of three independent experiments.
(B) One hour after TNFα stimulation, cells were collected for RNA isolation and subsequent reverse transcription. The relative mRNA levels of MCP-1, KC, and IL-6 in phosphatase RNAi transfected astrocytes after TNFα stimulation. The dashed lines represent a 3-fold increase and 70% reduction, respectively. Error bars represent the SD of three independent experiments.
(C) Summary of phosphatase interactions characterized in this report. NF-κB signaling was regulated by dephosphorylation of the TRAF2, IKK, and NF-κB complexes by the indicated PP2A cofactors.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2006 Elsevier Inc. Terms and Conditions